Enzymatic Assay of Phosphoglucomutase (EC 5.4.2.2)

Description

This procedure may be used for all Phosphoglucomutase products except for β-Phosphoglucomutase, Catalog Number P4109.

The continuous spectrophotometric rate detemination (A340, Light path = 1 cm) is based on the following reactions:

 

α-D-Glucose 1-Phosphate     Phosphoglucomutase   > α-D-Glucose 6-Phosphate

 

α-D-Glucose 6-Phosphate + β-NADP     G-6-PDH   > 6-Phospho-D-Gluconate + β-NADPH

β-NADP – β-Nicotinamide Adenine Dinucleotide Phosphate, Oxidized Form
β-NADPH – β-Nicotinamide Adenine Dinucleotide Phosphate, Reduced Form
G-6-PDH – Glucose-6-Phosphate Dehydrogenase

Unit Definition – One unit of phosphoglucomutase will convert 1.0 µmole of α-D-Glucose-1-Phosphate to α-D-Glucose-6-Phosphate per minute at pH 7.4 at 30 °C.

Precautions

Please consult the Safety Data Sheet for information regarding hazards and safe handling practices.

Reagents and Equipment Required

Gly-Gly (Catalog Number G1002)
α-D-Glucose 1-Phosphate, dipotassium salt, hydrate (Catalog Number G6875)
β-Nicotinamide Adenine Dinucleotide Phosphate, sodium salt, hydrate (Catalog Number N0505)
α-D-Glucose 1,6-bisphosphate, tetra(cyclohexylammonium) salt, hydrate (Catalog Number G5875)
1.00 M Magnesium Chloride Solution (Catalog Number M1028)
L-Cysteine hydrochloride, monohydrate (Catalog Number C7880)
Sodium bicarbonate (Catalog Number S8875)
Glucose-6-Phosphate Dehydrogenase from baker’s yeast (Catalog Number G6378 or G4134)
Cuvettes and thermostatted spectrophotometer

Preparation Instructions
(Storage/Stability)

Use ultrapure water (≥18 MΩ×cm resistivity at 25 °C) for the preparation of reagents.

Buffer (250 mM Glycylglycine Buffer, pH 7.4 at 30 °C) – Prepare a 33.04 mg/ml solution in ultrapure water using Gly-Gly (Catalog Number G1002). Adjust the pH of the solution to 7.4 at 30 °C.

G-1-P Solution (150 mM Glucose-1-Phosphate) – Prepare a 56.18 mg/ml solution in purified ultrapure using α-D-Glucose 1-Phosphate, dipotassium salt, hydrate (Catalog Number G6875).

β-NADP Solution (20 mM β-Nicotinamide Adenine Dinucleotide Phosphate) – Prepare a 16.7 mg/ml solution in ultrapure water using β-Nicotinamide Adenine Dinucleotide Phosphate, sodium salt, hydrate (Catalog Number N0505).

G-1,6-P Solution (23.2 mM Glucose-1,6-bisphosphate) – Prepare a 19 mg/ml solution in ultrapure water using α-D-Glucose 1,6-bisphosphate, tetra(cyclohexylammonium) salt, hydrate (Catalog Number G5875).

MgCl2 Solution (900 mM Magnesium Chloride) – Prepare a 0.90 ml/ml solution in ultrapure water using 1.00 M Magnesium chloride solution (Catalog Number M1028).

Cys Solution (260 mM L-Cysteine Hydrochloride) – Prepare a 45.65 mg/ml solution in ultrapure water using L-Cysteine hydrochloride, monohydrate (Catalog Number C7880). Adjust the pH of the solution to 7.0 using solid Sodium bicarbonate (Catalog Number S8875).

G-6-PDH Solution (100 units/ml, Glucose-6-Phosphate Dehydrogenase) – Immediately before use, prepare a solution containing 100 units/ml in cold Buffer using Glucose-6-Phosphate Dehydrogenase from baker’s yeast (Catalog Number G6378 or G4134).

Enzyme Solution (Phosphglucomutase) Immediately before use, prepare a solution containing 0.1–0.5 unit/ml of Phosphoglucomutase in cold Buffer.

Procedure

Final Assay Concentrations – In a 3.00 ml reaction mix, the final concentrations are 179 mM Glycylglycine, 5.0 mM Glucose 1-Phosphate, 0.67 mM β-Nicotinamide Adenine Dinucleotide Phosphate, 0.4 mM Glucose 1,6-bisphosphate, 30 mM Magnesium Chloride, 43 mM L-Cysteine, 1 unit of Glucose-6-Phosphate Dehydrogenase and 0.01–0.05 unit of Phosphoglucomutase.

1. Prepare a Reaction Cocktail by pipetting the following reagents into a suitable container:

 

Reagent Volume
(ml)
Buffer 40.80
G-1-P Solution 2.00
β-NADP Solution
2.00
G-1,6-P Solution 1.00
MgCl2 Solution 2.00
Cys Solution 10.00

 

2. Mix and equilibrate the Reaction Cocktail to 30 °C. Adjust the pH to 7.4 at 30 °C.

3. Pipette the following reagents into suitable cuvettes:

 

Reagent Volume (ml)
Test Blank
Reaction Cocktail 2.89 2.89
G-6-PDH Solution 0.01 0.01

 

4. Mix by inversion and equilibrate to 30 °C in a suitably thermostatted spectrophotometer. Monitor the A340 until constant. Then add:

Reagent Volume (ml)
Test Blank
Buffer
0.10
Enzyme Solution 0.10

 

5. Immediately mix by inversion and record the increase in A340 for ~5 minutes. Obtain the ΔA340/minute for both the blank and the test reactions using the maximum linear rate over a one minute interval using a minimum of 4 points.

Results

Calculations

1.

Units/ml enzyme = (ΔA340/minute Test – ΔA340/minute Blank) (3.00) (df)
(6.22) (0.10)

Where:

3.00 = Total volume (ml) of assay
df = Dilution Factor
6.22 = Millimolar extinction coefficient of β-NADPH at 340 nm
0.10 = Volume (ml) of Phosphoglucomutase used

 

2.

Units/mg protein = units/ml enzyme
mg protein/ml enzyme

Materials

     

References

  1. Bergmeyer, H.U. et al., Methods of Enzymatic Analysis, Bergmeyer, H.U., ed., Volume 1, 2nd ed., Academic Press, Inc., (New York, NY: 1974) 499-500.
  2. Lowry, O.H., and Passonneau., J.V., Journal of Biological Chemistry, 244, 910-916 (1969).

 

08/13-1

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